DESCRIPTION: (adapted from the abstract) The long-range objective of this proposal is to determine the roles of the pro-inflammatory cytokine, interleukin-1 (IL1) and its endogenous antagonist, IL1 receptor antagonist (IL1RA) in the physiological and pathophysiological modulation of vascular smooth muscle contraction and proliferation. Exogenous IL1 has potent effects on cultured human vascular smooth muscle cells (VSMC), inducing the synthesis of vasodilatory prostanoids acutely, and markedly stimulating human VSMC proliferation on longer-term exposure. During the previous grant period, human VSMC themselves were shown to produce both IL1-alpha precursor and an intracellular form of IL1RA (icIL1RA). Together these findings are suggestive of autocrine roles of VSMC-derived IL1 and IL1RA, but whether such autocrine effects exist has not been investigated. The proposed studies will test three hypotheses: 1) that IL1-alpha precursor and icIL1RA are autocrine regulators of VSMC proliferation; 2) that IL1-alpha precursor associates with the VSMC plasma membrane in a form which activates adjacent VSMC via direct cell-cell contact; and 3) that IL1-alpha precursor acts intracellularly to induce gene transcription via a mechanism dependent on its nuclear localization. Therefore there will be a determination: 1) Whether IL1-alpha precursor mediates the increased COX-2 mRNA in hypoxic human VSMC, and whether is effects are mediated intracellularly or via association with the plasma membrane; 2) Whether chronic overproduction of IL1-alpha precursor in VSMC stably transfected with IL1alpha precursor-encoding expression plasmids promote VSMC proliferation and increases the production of growth factors in VSMC; 3) Whether VSMC-derived icIL1RA inhibits VSMC proliferation, by stable transfecting of VSMC with sense and antisense expression plasmids to increase and inhibit icIL1RA production, respectively; 4) Whether IL1alpha precursor acts intracellularly within VSMC to activate IL1-inducible gene transcription or to activate IL1-inducible transcription factors; and 5) Whether the actions of IL1-alpha precursor in VSMC are dependent on its nuclear localization, by transfection VSMC with plasmids encoding a mutant Il1-alpha precursor deficient in nuclear localization. VSMC-derived Il1 may potentially contribute to multiple forms of human vascular disease: atherosclerotic VSMC proliferation, in which IL1-alpha mRNA has been demonstrated within medial VSMC of early lesions; VSMC responses to ischemia, as suggested by evidence that hypoxic VSMC produce IL1alpha; and septic shock, in which IL1 has been implicated as a key mediator, but the role of VSMC-derived IL1 is unknown. By elucidating the roles if IL1 and IL1RA in human VSMC, the proposed studies may significantly enhance our ability to control these major vascular complications of human disease.